This is the first competitive renewal of an innovative project at the interface of computational analysis of gene network regulation and behavioral pharmacology aimed at identifying and exploring new testable mechanistic and therapeutic hypotheses on the progression to excessive drinking and alcohol dependence, resilience and vulnerability to developing alcohol use disorder (AUD). The ultimate goal is to reveal and validate new and more effective therapeutic targets for AUD. Results of the current (first) funding period demonstrate that broad regulators of alcohol actions of phenotypic significance can be identified with the present experimental-computational systems biology strategies; which contributed to the identification of 3 candidate drugs for repositioning for AUD that are currently advancing toward clinical testing. Building on these results, in Specific Aim 1 of the proposed second funding period we aim to understand the molecular bases of the interactions of alcohol with the gene regulatory networks at a greater level of resolution by bringing to bear gene expression profiling of neurons, astrocytes, microglia and oligodendrocytes purified by fluorescence- activated cell sorting (FACS) from key brain regions of rats with histories of either moderate or excessive (escalated) alcohol self-administration. The sub-hypothesis under testing with this approach is that conducting gene regulation analyses at the cellular level of resolution will allow us to identify key cell type-specific and -common gene network dysregulations, and may point to previously unrecognized regulatory mechanisms of therapeutic potential. Specific Aim 2 will test the sub- hypothesis that the master regulator genes (MRs) governing the expression of specific gene signatures associated with the effects of alcohol have specific roles in motivation for alcohol and can serve as candidate druggable targets. In particular, studies in Specific Aim 2 will explore selected mechanistic hypotheses derived from gene regulatory analyses conducted in the current funding period and under the proposed Specific Aim 1 for their role in phenotypes of excessive alcohol drinking using computational, biochemical, behavioral and morphological strategies. Altogether, the present proposal will explore the transcriptional network dysregulations at the cell- type level of analysis associated with moderate and excessive alcohol intake to identify new mechanistic hypotheses on the neurobiological bases of excessive alcohol drinking that are expected to lead to the identification of novel therapeutic targets for AUD.